title: Development of a liquid chromatography/tandem mass spectrometry method to investigate the presence of biomarkers of DNA damage in urine related to red meat consumption and risk of colorectal cancer.
abstract: BACKGROUND
  The consumption of red meat is known to enhance the endogenous formation of N-nitroso compounds (NOCs), which are potent carcinogens. DNA damage related to NOCs, and hence red meat, has been detected in colorectal cells and in blood. We proposed to extend previous studies to a non-invasive approach for the detection of O(6)-carboxymethylguanine (O(6)CMG) and O(6)-carboxymethyl-2'-deoxyguanosine (O(6)CMdG) in urine in relation to red meat intake using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The presence of the adduct in urine samples either as the free base or as 2'-deoxynucleoside could help in determining the repair mechanism involved when such lesions are produced. A non-invasive assessment of DNA adducts could also allow for large-scale analyses in the population and cancer prevention dietary strategies.
  
  
  METHODS
  An LC/MS/MS method for the quantitation of O(6)CMG and O(6)CMdG was developed. Urine samples collected from healthy volunteers on red meat and vegetarian diets were analysed either by direct injection or after purification by solid-phase extraction (SPE). A separate LC/MS/MS method for O(6)-methylguanine (O(6)MeG) and O(6)-methyl-2'-deoxyguanosine (O(6)MedG), which are possible hydrolysis products forming during the sample pre-treatment, was also developed.
  
  
  RESULTS
  The developed LC/MS/MS method allowed the simultaneous measurement of O(6)CMG and O(6)CMdG. The limits of detection (LODs) were 0.38 ng/mL for O(6)CMG and 0.18 ng/mL for O(6)CMdG. The direct injection analysis of the clinical samples showed low sensitivity due to high background signal that was improved by SPE purification. However, the concentrations of the adducts in clinical samples were still found to be below the LOD.
  
  
  CONCLUSIONS
  Novel, reproducible, and accurate LC/MS/MS methods were developed for the determination of the urinary content of O(6)CMG and O(6)CMdG, and of the possible formation of O(6)MeG and O(6)MedG by decarboxylation. Clinical samples from volunteers on different diets were analysed. Further studies are required to discover a link between the presence of these biomarkers in urine and red meat consumption.
authors.affiliation: Department of Life, Health and Chemical Sciences, Faculty of Science, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK.
authors.forename: Chiara
authors.initials: C
authors.lastname: Da Pieve
authors.forename: Natasha
authors.initials: N
authors.lastname: Sahgal
authors.forename: Sharon A
authors.initials: SA
authors.lastname: Moore
authors.forename: Maria N
authors.initials: MN
authors.lastname: Velasco-Garcia
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D015179:Colorectal Neoplasms / Q000652:urine; D018592:Cross-Over Studies; D004249:DNA Damage; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000652:urine; D004032:Diet; D014676:Diet, Vegetarian; D006147:Guanine / Q000031:analogs & derivatives* / Q000652:urine; D006801:Humans; D057230:Limit of Detection; D008460:Meat / Q000032:analysis; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D006147:Guanine; C015558:O(6)-methyl-2'-deoxyguanosine; C008449:O-(6)-methylguanine; D003849:Deoxyguanosine